Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met₃₅–Val₃₆ as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.     

BRI2 protein regulates β-amyloid degradation by increasing levels of secreted insulin-degrading enzyme (IDE)

The amyloid precursor protein (APP) is one of the major proteins involved in Alzheimer disease (AD). Proteolytic cleavage of APP gives rise to amyloid-β (Aβ) peptides that aggregate and deposit extensively in the brain of AD patients. Although the increase in levels of aberrantly folded Aβ peptide is considered to be important to disease pathogenesis, the regulation of APP processing and Aβ metabolism is not fully understood. Recently, the British precursor protein (BRI2, ITM2B) has been implicated in influencing APP processing in cells and Aβ deposition in vivo. Here, we show that the wild type BRI2 protein reduces plaque load in an AD mouse model, similar to its disease-associated mutant form, ADan precursor protein (ADanPP), and analyze in more detail the mechanism of how BRI2 and ADanPP influence APP processing and Aβ metabolism. We find that overexpression of either BRI2 or ADanPP reduces extracellular Aβ by increasing levels of secreted insulin-degrading enzyme (IDE), a major Aβ-degrading protease. This effect is also observed with BRI2 lacking its C-terminal 23-amino acid peptide sequence. Our results suggest that BRI2 might act as a receptor protein that regulates IDE levels that in turn influences APP metabolism in a previously unrecognized way. Targeting the regulation of IDE may be a promising therapeutic approach to sporadic AD.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease

The secretion of proteins that lack a signal sequence to the extracellular milieu is regulated by their transition through the unconventional secretory pathway. IDE (insulin-degrading enzyme) is one of the major proteases of amyloid beta peptide (Aβ), a presumed causative molecule in Alzheimer disease (AD) pathogenesis. IDE acts in the extracellular space despite having no signal sequence, but the underlying mechanism of IDE secretion extracellularly is still unknown. In this study, we found that IDE levels were reduced in the cerebrospinal fluid (CSF) of patients with AD and in pathology-bearing AD-model mice. Since astrocytes are the main cell types for IDE secretion, astrocytes were treated with Aβ. Aβ increased the IDE levels in a time- and concentration-dependent manner. Moreover, IDE secretion was associated with an autophagy-based unconventional secretory pathway, and depended on the activity of RAB8A and GORASP (Golgi reassembly stacking protein). Finally, mice with global haploinsufficiency of an essential autophagy gene, showed decreased IDE levels in the CSF in response to an intracerebroventricular (i.c.v.) injection of Aβ. These results indicate that IDE is secreted from astrocytes through an autophagy-based unconventional secretory pathway in AD conditions, and that the regulation of autophagy is a potential therapeutic target in addressing Aβ pathology.     

Reduced amyloid‐β degradation in early Alzheimer's disease but not in the APPswePS1dE9 and 3xTg‐AD mouse models

Alzheimer's disease (AD) is hallmarked by amyloid‐β (Aβ) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Aβ can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg‐AD mouse models. A quenched Aβ peptide that becomes fluorescent upon degradation was used to screen for Aβ‐degrading cytoplasmic peptidases cleaving the aggregation‐prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Aβ‐degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg‐AD mice. Insulin‐degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Aβ. Oligomerization of Aβ prevents its clearance by IDE. Intriguingly, the Aβ‐degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg‐AD mouse models do not show altered Aβ degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Aβ peptides are representative for human sAD.     

Leptin inhibits amyloid β-protein degradation through decrease of neprilysin expression in primary cultured astrocytes

Pathogenesis of Alzheimer’s disease (AD) is characterized by accumulation of extracellular deposits of amyloid β-protein (Aβ) in the brain. The steady state level of Aβ in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aβ deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aβ degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aβ in primary cultured astrocytes. These results suggest that leptin suppresses Aβ degradation by NEP through activation of ERK.     

Antagonist of peroxisome proliferator-activated receptor γ induces cerebellar amyloid-β levels and motor dysfunction in APP/PS1 transgenic mice

Recent evidences show that peroxisome proliferator-activated receptor γ (PPARγ) is involved in the modulation of the amyloid-β (Aβ) cascade causing Alzheimer’s disease (AD) and treatment with PPARγ agonists protects against AD pathology. However, the function of PPARγ steady-state activity in Aβ cascade and AD pathology remains unclear. In this study, an antagonist of PPARγ, GW9662, was injected into the fourth ventricle of APP/PS1 transgenic mice to inhibit PPARγ activity in cerebellum. The results show that inhibition of PPARγ significantly induced Aβ levels in cerebellum and caused cerebellar motor dysfunction in APP/PS1 transgenic mice. Moreover, GW9662 treatment markedly decreased the cerebellar levels of insulin-degrading enzyme (IDE), which is responsible for the cellular degradation of Aβ. Since cerebellum is spared from significant Aβ accumulation and neurotoxicity in AD patients and animal models, these findings suggest a crucial role of PPARγ steady-state activity in protection of cerebellum against AD pathology.     

The irreversible binding of amyloid peptide substrates to insulin-degrading enzyme

Insulin-degrading enzyme (IDE) is a conserved Zn2+metalloendopeptidase involved in insulin degradation and in the maintenance of brain steady-state levels of amyloid β peptide (Aβ) of Alzheimer’s disease (AD). Our recent demonstration that IDE and Aβ are capable of forming a stoichiometric and extremely stable complex raises several intriguing possibilities regarding the role of this unique protein-peptide interaction in physiological and pathological conditions. These include a protective cellular function of IDE as a “dead-end chaperone” alternative to its proteolytic activity and the potential impact of the irreversible binding of Aβ to IDE upon its role as a varicella zoster virus receptor. In a pathological context, the implications for insulin signaling and its relationship to AD pathogenesis are discussed. Moreover, our findings warrant further research regarding a possible general and novel interaction between amyloidogenic peptides and other Zn2+metallopeptidases with an IDE-like fold and a substrate conformation-dependent recognition mechanism.     

Degradation of Alzheimer’s Amyloid-β by a Catalytically Inactive Insulin-Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer’s amyloid-β (Aβ). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aβ degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aβ(1–40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aβ(1–40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aβ fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aβ(1–40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aβ(1–40) inactivates cf-E111Q-IDE’s catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aβ degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE’s function.     

New Insights into Epigenetic and Pharmacological Regulation of Amyloid-Degrading Enzymes

Currently, deficit of amyloid β-peptide (Aβ) clearance from the brain is considered as one of the possible causes of amyloid accumulation and neuronal death in the sporadic form of Alzheimer’s disease (AD). Aβ clearance can involve either specific proteases present in the brain or Aβ-binding/transport proteins. Among amyloid-degrading enzymes the most intensively studied are neprilysin (NEP) and insulin-degrading enzyme (IDE). Since ageing and development of brain pathologies is often accompanied by a deficit in the levels of expression and activity of these enzymes in the brain, there is an urgent need to understand the mechanisms involved in their regulation. We have recently reported that NEP and also an Aβ-transport protein, transthyretin are epigenetically co-regulated by the APP intracellular domain (AICD) and this regulation depends on the cell type and APP₆₉₅ isoform expression in a process that can be regulated by the tyrosine kinase inhibitor, Gleevec. We have now extended our work and shown that, unlike NEP, another amyloid-degrading enzyme, IDE, is not related to over-expression of APP₆₉₅ in neuroblastoma SH-SY5Y cells but is up-regulated by APP₇₅₁ and APP₇₇₀ isoforms independently of AICD but correlating with reduced HDAC1 binding to its promoter. Studying the effect of the nuclear retinoid X receptor agonist, bexarotene, on NEP and IDE expression, we have found that both enzymes can be up-regulated by this compound but this mechanism is not APP-isoform specific and does not involve AICD but, on the contrary, affects HDAC1 occupancy on the NEP gene promoter. These new insights into the mechanisms of NEP and IDE regulation suggest possible pharmacological targets in developing AD therapies.     

Somatostatin in Alzheimer's disease: A new Role for an Old Player

The amyloid beta (Aβ) peptide is central to the pathogenesis of Alzheimer's disease (AD). Insights into Aβ-interacting proteins are critical for understanding the molecular mechanisms underlying Aβ-mediated toxicity. We recently undertook an in-depth in vitro interrogation of the Aβ1–42 interactome using human frontal lobes as the biological source material and taking advantage of advances in mass spectrometry performance characteristics. These analyses uncovered the small cyclic neuropeptide somatostatin (SST) to be the most selectively enriched binder to oligomeric Aβ1–42. Subsequent validation experiments revealed that SST interferes with Aβ fibrillization and promotes the formation of Aβ assemblies characterized by a 50–60 kDa SDS-resistant core. The distributions of SST and Aβ overlap in the brain and SST has been linked to AD by several additional observations. This perspective summarizes this body of literature and draws attention to the fact that SST is one of several neuropeptide hormones that acquire amyloid properties before their synaptic release. The latter places the interaction between SST and Aβ among an increasing number of observations that attest to the ability of amyloidogenic proteins to influence each other. A model is presented which attempts to reconcile existing data on the involvement of SST in the AD etiology.     

Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway

Background

Insulin degrading enzyme (IDE) is a major protease of amyloid beta peptide (Aβ), a prominent toxic protein in Alzheimer’s disease (AD) pathogenesis. Previous studies suggested that statins promote IDE secretion; however, the underlying mechanism is unknown, as IDE has no signal sequence.

Results

In this study, we found that simvastatin (0.2 μM for 12 h) induced the degradation of extracellular Aβ₄₀, which depended on IDE secretion from primary astrocytes. In addition, simvastatin increased IDE secretion from astrocytes in a time- and dose-dependent manner. Moreover, simvastatin-mediated IDE secretion was mediated by an autophagy-based unconventional secretory pathway, and autophagic flux regulated simvastatin-mediated IDE secretion. Finally, simvastatin activated autophagy via the LKB1-AMPK-mTOR signaling pathway in astrocytes.

Conclusions

These results demonstrate a novel pathway for statin-mediated IDE secretion from astrocytes. Modulation of this pathway could provide a potential therapeutic target for treatment of Aβ pathology by enhancing extracellular clearance of Aβ.

     

综述:中性内肽酶在阿尔茨海默病发病机制中的作用

β-淀粉样蛋白(β amyloid,Aβ)在海马区的沉积是阿尔茨海默病(Alzheimer′s disease,AD)发病的典型表现,清除或降低Aβ含量是治疗AD的目标之一．较之Aβ生成的增多,体内降解Aβ能力的下降在AD发病过程中显得更为重要．尽管Aβ在体内可以通过运输到血液和脑脊液途径来清除,但大部分Aβ被中性内肽酶(neprilysin,NEP)为代表的一类蛋白酶降解为小分子后从体内清除．老年人、轻度认知障碍期(MCI)和AD患者的NEP活性显著下降,且NEP活性下降与脑内Aβ升高及AD患者认知功能损伤相关．NEP有可能成为AD治疗的潜在药物靶点,针对轻度认知障碍前期(pre-MCI)和MCI,提高NEP的活性,促进Aβ的降解,有可能延缓AD的发生和发展．     

Treadmill Exercise Ameliorates Spatial Learning and Memory Deficits Through Improving the Clearance of Peripheral and Central Amyloid-Beta Levels

Aggregated amyloid beta (Aβ) peptides are believed to play a decisive role in the pathology of Alzheimer’s disease (AD). Previous evidence suggested that exercise contributes to the improvement of cognitive decline and slows down pathogenesis of AD; however, the exact mechanisms for this have not been fully understood. Here, we evaluated the effect of a 4-week moderate treadmill exercise on spatial memory via central and peripheral Aβ clearance mechanisms following developed AD-like neuropathology induced by intra-hippocampal Aβ_1–42 injection in male Wistar rats. We found Aβ_1–42-treated animals showed spatial learning and memory impairment which was accompanied by increased levels of amyloid plaque load and soluble Aβ_1–42 (sAβ_1–42), decreased mRNA and protein expression of neprilysin (NEP), insulin degrading enzyme (IDE) and low-density lipoprotein receptor-related protein-1 (LRP-1) in the hippocampus. Aβ_1–42-treated animals also exhibited a higher level of sAβ_1–42 and a lower level of soluble LRP-1 (sLRP-1) in plasma, as well as a decreased level of LRP-1 mRNA and protein content in the liver. However, exercise training improved the spatial learning and memory deficits, reduced both plaque load and sAβ_1–42 levels, and up-regulated expression of NEP, IDE, and LRP-1 in the hippocampus of Aβ_1–42-treated animals. Aβ_1–42-treated animals subjected to treadmill exercise also revealed decreased levels of sAβ_1–42 and increased levels of sLRP-1 in plasma, as well as increased levels of LRP-1 mRNA and protein in the liver. In conclusion, our findings suggest that exercise-induced improvement in both of central and peripheral Aβ clearance are likely involved in ameliorating spatial learning and memory deficits in an animal model of AD. Future studies need to determine their relative contribution.     

High-molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Alzheimer's disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ(42) -positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ(40) - and Aβ(42) preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ(40) and strikingly reduced levels of hiMWAβ(42) . These results indicate that hiMWAβ aggregates, particularly Aβ(42) species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.     

β-淀粉样蛋白-血红素复合物和蛋白质酪氨酸硝化及其与阿尔茨海默症的关系

β-淀粉样蛋白(Amyloid-β,Aβ)是阿尔茨海默症(Alzheimer’s disease,AD)病人大脑中淀粉样斑块的主要组成部分。β-淀粉样蛋白级联假说指出,Aβ在脑实质的沉积是最终导致阿尔茨海默症的一个关键步骤。目前的大量研究表明,相对于高度聚集的Aβ,可溶性的Aβ低聚物可能与认知功能障碍的关联性更强。血红素(heme)的代谢在AD患者大脑中发生了改变。近来发现heme可与Aβ结合,形成一个复合物Aβ-heme,该复合物拥有显著高于heme的过氧化物酶活性,具有比heme更强的催化蛋白质酪氨酸硝化的能力。这个结果提示,Aβ-heme可能是联系Aβ与AD中大量蛋白质发生硝化的关键分子。同时,Aβ与heme的结合改变了heme催化蛋白质硝化的位点选择性。这些研究对于阐明Aβ和heme在体内可能的生理作用具有重要意义。     

Comparative studies for amyloid beta degradation: “Neprilysin vs insulysin”, “monomeric vs aggregate”, and “whole Aβ40 vs its peptide fragments”

Amyloid beta (Aβ) proteins are produced from amyloid precursor protein cleaved by β- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aβs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aβs in vivo, with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aβ₄₀, aggregated Aβ₄₀, and Aβ₄₀ peptide fragments, including Aβ₁₆. Our results allowed identification of all the peptide fragments from non-aggregated Aβ₄₀: NEP, 23 peptide fragments consisting of 2–11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6–33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aβ₄₀, whereas NEP can digest both Aβ₄₀ and partial structures such as Aβ₁₆ and peptide fragments generated by the digestion of Aβ₄₀ by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aβ₄₀.     

Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations

The amyloid β-peptide (Aβ), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid β-protein precursor (APP) through consecutive proteolysis by β- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aβ production and increase the ratio of the aggregation-prone 42-residue form (Aβ42) to the 40-residue form (Aβ40). The connection between these two effects is not understood. Besides Aβ40 and Aβ42, γ-secretase produces a range of Aβ peptides, the result of initial cutting at the ε site to form Aβ48 or Aβ49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aβ peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aβ peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aβ42:Aβ40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aβ peptides.